Trends in Synthesis and Manufacturing

!--h2>

Diana Gitig, Ph.D.

Peptides are popular, and these days researchers want more and more from them. They want increasingly complex peptides, they want high-quality peptides that conform to cGMP, and they want larger and larger quantities of pure peptides. And they want them fast. Different companies have various takes on where the peptide-synthesis market is, where it is heading, how to meet current demands, and what to do to capitalize on the trends.

Researchers want peptides primarily for drugs. This is true for scientists in government and academic laboratories, as much as it is for those in biotech and pharma labs. Peptide drugs are currently in all phases of clinical trials.

“Peptides are ideal candidates for treating niche diseases,” remarks Rodney Lax, the director of sales and marketing at PolyPeptide Laboratories (www.polypeptide.com). “Of course, they will almost certainly also be recruited in the fights against cancer as well as metabolic and neurodegenerative diseases.”

Peptides have only recently been exploited as drug candidates, since previously, no one thought that they could be effective drugs, according to Anita Ho, Ph.D., president of Anaspec (www.anaspec.com). Synthetic amylin, which has been used for appetite suppression and to treat insulin-dependent diabetes has changed all of that. Remarkably, synthetic amylin is the first drug to be approved by the FDA to lower blood sugar in type 1 diabetics since the discovery of insulin in the early 1920s.

American Peptide Company (www.americanpeptide.com) in collaboration with R&D Bioproducts (www.rndbioproducts.com) has developed a peptide that it says has significant therapeutic potential to be used against ubiquitin-related neurodegenerative disorders including Alzheimer’s, Huntington’s, and Parkinson’s diseases as well as amyotrophic lateral sclerosis.

The drug reportedly mimics the binding site of TRAF-6, an E3 ubiquitin ligase, and thus prevents the ubiquitination and degradation of those signaling enzymes usually targeted by the full length TRAF-6.

Labeled Peptides

In addition to peptide drugs, Dr. Ho reports that many customers are ordering labeled peptides. FRET studies use a peptide linked to a fluorescent donor along with a quencher as substrates for proteases. “Isotope-labeled peptides will be the next big thing,” Dr. Ho insists, “since many unknown proteins are currently being isolated, and labeled-protein fragments will be required for comparisons to tryptic digests.”

Driving Factors

Patricia Haller, Ph.D., and Jianjun Jiang, Ph.D., the director and vp of American Peptide’s GMP manufacturing division, respectively, point out that the growth and maturation of the drug delivery market has facilitated the use of peptides as drugs and promoted their efficacy.

“The small molecule drug market is saturated,” Dr. Haller says. “Peptides are preferable to small molecules because they are more natural and thus have lower toxicity. But they have a short half life. New delivery vectors have allowed peptides to be effective drugs in the commercial market. They are becoming more user friendly, for example, they might require an inhaler rather than an injection or one injection per day rather than three.”

According to Dr. Ho, some other factors driving the peptide-synthesis market include proteomics—isotopically labeled peptides will increasingly be used to identify unknown proteins—and antibody production, as using just your desired epitope is quicker and easier than using a whole protein.

PolyPeptide Laboratories is seeing “a healthy growth in the field of generics,” Lax says. “Peptide generics are resilient.” This is important since patents are constantly expiring. He also lists decreasing costs of manufacture due to lower raw material prices, improved separation technology, and better understanding of peptide drug design as positively impacting the peptide-synthesis market.

Expansion of Manufacturing Capabilities

American Peptide is committed to exceeding its capacity to meet the demand for quality peptides, explains Dr. Jiang. The company is doubling its production line and increasing the size of its California facility. It is also in the process of building a 200-liter lyophilizer, which will be the largest in the U.S. for peptide manufacturing, according to Dr. Jiang.

When dealing with academic researchers, American Peptide assists in selecting sequences but is careful not to dictate them, Dr. Jiang says. It gives data on solubility and aggregation potentials before cGMP are put into effect; this strong foundation at the preclinical stage saves time and money later, he reports.

PolyPeptide Laboratories is also expanding; it purchased the NeoMPS group in November. Lax notes that this acquisition broadened the company’s range of peptide services in addition to giving it additional physical space to grow into. It was already operating in Malmö, Sweden, in Torrance, CA, and at a new 35,000 sq. ft. plant near Mumbai, India. With the addition of NeoMPS, facilities have been added in Strasbourg, France, and San Diego.

The chemistry application team at Protein Technologies(www.peptideinstruments.com) has made some technical strides recently. HCTU is an aluminum salt coupling reagent that the team claims is “extremely efficient, yet affordable.” Protein Technologies has used it “to perform fast syntheses of even difficult and long peptides such as beta-amyloid and a 68-mer chemokine.” Coupling times of five minutes or less and deprotection times of three minutes or less using HCTU as the activator and conventional chemistry were achieved.

Technical Strides

Beta-amyloid (1-42) was synthesized on Protein Technologies’ Symphony® peptide synthesizer, utilizing its 12 independent reaction vessels. Three resins, two activators, and three reaction times can be tested in the same synthesis, since different programs can be run on different reaction vessels simultaneously, reports Christina A. Chantell, a chemist at Protein Technologies.

Synthesis was performed on Fmoc-Ala-Wang ChemMatrix resin (0.56 mmol/g), Fmoc-Ala-HMPB ChemMatrix resin (0.54 mmol/g), and Fmoc-Ala-Wang PS LL resin (0.25 mmol/g) using HCTU in 30 hours or 54 hours total synthesis time, notes Chantell. Using HCTU/HOCt or HATU, total synthesis time was 12 hours, she adds.

According to Chantell, Beta-amyloid (1-42) could be produced in 12 hours with deprotection times of 2 x 1 minute and a coupling time of five minutes, such that it could be purified successfully by analytical HPLC. The resulting peptide identity was verified by mass spectrometry. Using Fmoc-Ala-Wang PS LL resin and HCTU produced the peptide for the least cost, she adds.